Wnt proteins form a family of highly conserved secreted signaling molecules that regulate cell-to-cell interactions during embryogenesis. Wnt genes and Wnt signaling are also implicated in cancer. Insights into the mechanisms of Wnt action have emerged from several systems: genetics in Drosophila and Caenorhabditis elegans; biochemistry in cell culture and ectopic gene expression in Xenopus embryos. Many Wnt genes in the mouse have been mutated, leading to very specific developmental defects. As currently understood, Wnt proteins bind to receptors of the Frizzled family on the cell surface. Through several cytoplasmic relay components, the signal is transduced to beta-catenin, which then enters the nucleus and forms a complex with TCF to activate transcription of Wnt target genes. Expression of Wnt proteins varies, but is often associated with developmental process, for example in embryonic and fetal tissues.
The exploration of physiologic functions of Wnt proteins in adult organisms has been hampered by functional redundancy and the necessity for conditional inactivation strategies. Dickkopf-1 (Dkk1) has been recently identified as the founding member of a family of secreted proteins that potently antagonize Wnt signaling (see Glinka et al. (1998) Nature 391:357-62; Fedi et al. (1999) J Biol Chem 274:19465-72; and Bafico et al. (2001) Nat Cell Biol 3:683-6).
Signaling in the pathway is believed to be initiated by the secreted wnt proteins, which bind to a class of seven-pass transmembrane receptors encoded by the frizzled genes. Activation of the receptor leads to the phosphorylation of the disheveled protein which, through its association with axin, prevents glycogen synthase kinase 3beta (GSK3beta) from phosphorylating critical substrates. In vertebrates, the inactivation of GSK3beta might result from its interaction with Frat-1. The GSK3beta substrates include the negative regulators axin and APC, as well as β-catenin itself. Unphosphorylated β-catenin escapes recognition by β-TRCP, a component of an E3 ubiquitin ligase, and translocates to the nucleus where it engages transcription factors such as TCF and LEF. Additional components in the pathway include casein kinases I and II, both of which have been proposed to phosphorylate disheveled. The serine/threonine phosphatase PP2A associates with axin and APC. In the absence of wnt, GSK3beta phosphorylates APC and axin, increasing their binding affinities for β-catenin, which too is phosphorylated by GSK3beta, marking it for destruction. In the presence of wnt, FRAT prevents GSK3beta from phosphorylating its substrates, and β-catenin is stabilized. Casein kinase1epsilon (CK1epsilon) binds to and phosphorylates disheveled (dvl), modulating the FRAT1/GSK3beta interaction.
The wnt ligands, of which there are at least 16 members in vertebrates, are secreted glycoproteins that can be loosely categorized according to their ability to promote neoplastic transformation. There are also numerous wnt receptors. At least 11 vertebrate frizzled genes have been identified. In addition to the frizzled receptors, there exists a family of secreted proteins bearing homology to the extracellular cysteine-rich domain of frizzled. The so-called secreted frizzled-related proteins (sFRP) bind to the wnt ligands, thereby exerting antagonistic activity when overexpressed in wnt signaling assays. The vertebrate sFRPs, like the frizzled proteins, exhibit functional specificity with respect to the various wnts.
Mutations in several genes are associated with tumorigenicity, including b-catenin, APC and Axin. Mutations in the β-catenin gene (CTNNb1) affecting the amino-terminal region of the protein make it refractory to regulation by APC. These mutations affect specific serine and threonine residues, and amino acids adjacent to them, that are essential for the targeted degradation of β-catenin. These regulatory sequence in β-catenin are mutated in a wide variety of human cancers as well as in chemically and genetically induced animal tumors. Axin is regarded as a tumor suppressor, which when mutated alters the Wnt signaling pathway.
APC is a tumor suppressor in human cancers and its mutation relates strongly to the regulation of β-catenin. The spectrum of APC mutations, which typically truncate the protein, suggest selection against β-catenin regulatory domains, albeit not necessarily against β-catenin binding. The presence of axin binding sites are critical to APC in the regulation of beta-catenin levels and signaling in cultured cells. In colorectal cancer, the vast majority of tumors contain APC mutations, although the overall frequency of β-catenin mutations is quite low. When colorectal tumors lacking APC mutations were analyzed separately, the likelihood of finding a CTNNb1 mutation was greatly increased.
Aggressive fibromatosis, otherwise known as desmoid tumor, is a locally invasive fibrocytic growth that occurs with increased incidence in patients with familial adenomatous polyposis coli (FAP). FAP individuals carry APC mutations in their germline and present with multiple intestinal adenomas at an early age. Desmoids also occur sporadically and, with the exception of colorectal cancer, represent a rare example of biallelic inactivation of APC in individuals without a pre-existing germline mutation in APC. Mutations in CTNNb1 have also been detected in sporadic desmoid tumors.
Several mutations in CTNNb1 were recently identified in gastric cancers, which occur with increased incidence in FAP patients. In one study, 27% of intestinal type gastric cancers harbored mutations in β-catenin. Hepatoblastoma also occurs with increased incidence in FAP individuals. In three separate studies, mutations in β-catenin were identified at high frequency in hepatoblastoma. Thyroid cancers also occur with increased incidence in FAP and a high frequency of CTNNb1 mutations has been reported for anaplastic thyroid cancers. Hepatocellular carcinoma (HCC) is one of the most common tumors harboring mutations in the wnt pathway. The frequency of CTNNb1 mutations in hepatocellular carcinoma (HCC) was ˜20% overall and may be higher for HCCs associated with hepatitis C virus. Some cancers, such as endometrial ovarian tumors, do not occur with increased incidence in patients with FAP, yet they contain activating mutations in CTNNb1. The CTNNb1 mutations associated with ovarian cancer appeared to be confined to the endometrioid subtype. Additional types of cancers with CTNNb1 mutations include melanoma and prostate. The highest percentage of CTNNb1 mutations occurs in a common skin tumor known as pilomatricomas.
Conventional cytotoxic chemotherapy for cancer targets rapidly dividing cells within tumors. Correspondingly, such chemotherapy is generally limited by its effects on rapidly dividing cells in normal tissues in patients, such as those in the hematopoietic system, the lining of the gastrointestinal tract, and the skin.
In another example, ionizing radiation (IR) is used to treat about 60% of cancer patients, by depositing energy that injures or destroys cells in the area being treated. Radiation injury to cells is nonspecific, with complex effects on DNA. The efficacy of therapy depends on cellular injury to cancer cells being greater than to normal cells. Radiotherapy may be used to treat every type of cancer. Some types of radiation therapy involve photons, such as X-rays or gamma rays. Another technique for delivering radiation to cancer cells is internal radiotherapy, which places radioactive implants directly in a tumor or body cavity so that the radiation dose is concentrated in a small area.
Radiotherapy may be used in combination with additional agents. Radiosensitizers make the tumor cells more likely to be damaged, and radioprotectors protect normal tissues from the effects of radiation. Hyperthermia is also being studied for its effectiveness in sensitizing tissue to radiation.
A method to prevent toxicity to normal tissues while preserving efficacy against tumor cells would augment the therapeutic index of chemotherapy and radiation therapy and limit the adverse side effects of such treatments for patients. In addition, the availability of this type of technology would permit the safe use of higher doses of therapy with enhanced anti-tumor effects. At present effective methods of this type have not been described and none have entered widespread clinical use. The present invention is designed to meet this need.
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Wnts act by binding the receptors of the Frizzled family (Bhanot et al. (1996) Nature 382:225-30) in association with the low-density lipoprotein receptor related proteins (LRP). In the absence of a Wnt signal, the serine/threonine kinase GSK-3β phosphorylates beta-catenin, targeting it for ubiquitination and degradation by proteosomes. Binding of Wnt proteins to their receptors leads to beta-catenin stabilization and accumulation in the cytosol (Willert & Nusse (1998) Curr Opin in Gen Dev 8:95-102). Beta-catenin can then translocate to the nucleus, where it binds to members of the LEF-1/TCF family of transcription factors and causes induction of target genes Eastman & Grosschedl (1999) Curr Opin Cell Biol 11:233-40).
The use of β-catenin in the expansion of stem cells is discussed in U.S. Pat. No. 6,465,249. The use of wnt to stimulate hematopoietic stem cells is proposed in U.S. Pat. No. 5,851,984. Protection is stem cells is discussed in US-2004-0171559-A1.